One of the most serious problems that faces a practicing oncologist is the metastasis of malignant tumor cells from the primary site to multiple, distant sites. In most cases of cancer, it is the metastatic lesions that kill the patient. While surgery is often effective against a primary tumor, it cannot excise all malignant tissue if the cancer has metastasized. For example, one-third of patients with operable breast cancer develop metastases after primary therapy. Adjuvant therapy can improve this prognosis, but requires identification of high-risk patients. Staging primary tumor by size and axillary nodes status is insufficient for this purpose.
Bone and bone marrow are frequent sites of metastasis, but radiologic and scintographic techniques can detect bone involvement only when destruction of bone matrix has occurred. The presence of epithelial tumor cells in bone marrow generally correlates with conventional risk factors, such as size and histological grade of the primary carcinoma, distant metastasis and lymph node involvement. Clinical follow-up studies have demonstrated a significantly increased relapse rate in patients that presented with epithelial tumor cells in bone marrow at the time of primary surgery.
Methods are needed that allow the detection of disseminated tumor cells at a time when they have not yet developed into incurable
Methods are needed that allow the detection of disseminated tumor cells at a time when they have not yet developed into incurable macrometastases. Such a diagnosis could be helpful in determining prognosis, in deciding whether a particular therapy is indicated, and in providing a way to monitor the effectiveness of therapy. Such a diagnosis might utilize the different morphology of tumor cells, as compared to normal cells of the bone and blood. Tumor specific antigens on the cell surface may be detected. Where the tumor cell is of a different developmental lineage than hematopoietic cells, for example epithelial carcinomas, then tissue specific markers may be used to identify the tumor cells.
Methods have been described for detection of spreading cancer cells into bone marrow, peripheral blood and secondary lymphoid organs from small localized primary tumors. Morphologic analysis may be performed by cytospin preparations of bone marrow smears, peripheral blood or lymph node cell samples, followed by May Grunwald-Giemsa staining and examination by light microscopy (Molino et al., 1991). Alternatively, cytospin preparations, or smears of cells, may also be stained with tumor specific or tissue specific antibodies. These methods suffer from extremely low sensitivity, and are time-consuming and laborious. As many as 100 slides might have to be examined to detect the presence of a single tumor cell.
Disseminated tumor cells have also been detected through the use of reverse transcriptase and polymerase chain reaction (RT-PCR). PCR may be used to amplify prostate specific antigen (PSA) mRNA or cytokeratin 19 mRNA. These methods have several disadvantages, particularly with respect to low sensitivity and false-positive results.
It has been shown that breast carcinoma cells can be identified or isolated from a peripheral blood sample by fluorescence activated cell sorting (Gross et al., 1995). However, the high technological effort required for FACS is a barrier to its routine use in medical diagnosis. FACS analysis or sorting is a time consuming and cost intensive procedure. Flow cytometry has the additional disadvantage in that it is difficult to sort or analyze large numbers of cells, or multiple samples at the same time.
An alternative approach to cell sorting has been described, whereby magnetic microparticles coupled to antibodies are used to select for specific cell types. Shpall et al., (1991) have described a method and a device for immunomagnetic purging of breast cancer cells from bone marrow cell samples for autologous transplantation of carcinoma patients receiving high-dose chemotherapy.
An improved magnetic sorting process whereby tumor cells could be separated from peripheral blood or other tissue sources, and which allows multiple, samples to be run on the bench would provide numerous benefits to the field of oncologic diagnosis.
Relevant Literature
Detection of disseminated tumor cells by morphology on cytospin preparations or blood smears is described in Molino et al. (1991) Cancer 67:1033. Use of the same technique in conjunction with antibody staining for tissue or tumor specific antigens is described in Redding et al. (1983) The Lancet 3:1271; Schlimok et al. (1987) P.N.A.S. 84:8672; Moul et al. (1994) Urology 43:68; Menard et al. (1994) Br. J. Cancer; Osborne et al. (1991) Cancer Res. 51:2706; Cote et al. (1991) J. Clin. Oncol. 9:1749; Bretton et al. (1994) The Prostate 25:108; and Oberneder et al. (1994) Urol. Res. 22:3.
Use of reverse transcriptase and the polymerase chain reaction to detect expression of tumor or tissue specific genes from samples of peripheral blood, bone marrow or lymph nodes is described in Seiden et al. (1994) J. Clin. Oncol. 12:2634; Katz et al. (1994) Urology 43:765; Schoenfeld et al. (1994) Cancer Res. 54:2986; and Datta et al. (1994) J. Clin. Oncol. 12:475.
Detection and separation of tumor cells from peripheral blood by flow cytometry is described in Gross et al. (1995) P.N.A.S. 92:537. Methods utilizing immunomagnetic separations are described in Shpall et al. (1991) Bone Marrow Transplantation 7:145; Kemmner et al. (1992) J. Immunol. Methods 147:197; and Griwatz et al. (1994) Suppl. J. Exp. Clin. Cancer Res. 13 No. 3 (Abstract).
Correlations between the presence of disseminated tumor cells in hematopoietic organs and conventional risk factors are noted in Schlimok et al. (1991) Eur. J. Cancer 27:1461; Huvos et al. (1971) Ann. Surg. 173:44; International (Ludwig) breast cancer study group (1990) Lancet 335:1565; and DeMascarel et al. (1992) Br. J. Cancer 66:523.
High gradient magnetic cell sorting is described in Miltenyi et al. (1990) Cytometry 11:231-238. Molday, U.S. Pat. No. 4,452,773 describes the preparation of magnetic iron-dextran microspheres and provides a summary describing the various means of preparation of particles suitable for attachment to biological materials. A description of polymeric coatings for magnetic particles used in HGMS are found in DE 3720844 (Miltenyi) and Miltenyi et al., U.S. Pat. No. 5,385,707. Methods to prepare superparamagnetic particles are described in U.S. Pat. No. 4,770,183.